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Review
. 2024 Jan;19(1):3-29.
doi: 10.1038/s41596-023-00910-y. Epub 2023 Nov 14.

Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii

Nadege Philippe #  1 , Avi Shukla  1 , Chantal Abergel  2 , Hugo Bisio #  3
Affiliations
Free article
Review

Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii

Nadege Philippe et al. Nat Protoc. 2024 Jan.
Free article

Abstract

Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances. Their host, Acanthamoeba castellanii, is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host were lacking. Here, we provide a detailed method for genetic manipulation of A. castellanii and the GVs it plays host to by using CRISPR-Cas9 or homologous recombination. We detail the steps of vector preparation (4 d), transfection of amoeba cells (1 h), infection (1 h), selection (5 d for viruses, 2 weeks for amoebas) and cloning of recombinant viruses (4 d) or amoebas (2 weeks). This procedure takes ~3 weeks or 1 month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 h), western blotting (4 h), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 d) and quantification of DNA replication by quantitative PCR (4 h) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen or pathogen-symbiont interactions in A. castellanii, to be studied in vivo.

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References

    1. Rayamajhee, B., Willcox, M. D., Henriquez, F. L., Petsoglou, C. & Carnt, N. Acanthamoeba keratitis: an increasingly common infectious disease of the cornea. Lancet Microbe 2, e345–e346 (2021). - DOI - PubMed
    1. Guimaraes, A. J., Gomes, K. X., Cortines, J. R., Peralta, J. M. & Peralta, R. H. Acanthamoeba spp. as a universal host for pathogenic microorganisms: one bridge from environment to host virulence. Microbiol. Res. 193, 30–38 (2016). - DOI - PubMed
    1. Raoult, D. et al. The 1.2-megabase genome sequence of Mimivirus. Science 306, 1344–1350 (2004). - DOI - PubMed
    1. Philippe, N. et al. Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science 341, 281–286 (2013). - DOI - PubMed
    1. Legendre, M. et al. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proc. Natl Acad. Sci. USA 111, 4274–4279 (2014). - DOI - PubMed - PMC

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